Method for improving affinity of antibody for antigen and its use

Abstract

To provide a novel method for improving affinity to an antigen of an antibody by modifying an amino acid residue in a heavy chain framework region without changing an amino acid sequence of a complementarity determining region, and to provide a novel antibody with improved affinity for the antigen.SOLUTION: The above problem is solved by making at least three selected from amino acid residues at position 68, 70, 72, 74, 75, 77, 79, 81, 82A, 82B, 83, 84, 85 and 87 of a heavy chain defined by the Kabat method an arginine residue or a lysine residue, in an amino acid sequence of a framework region 3 of the heavy chain of an antibody.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a method for improving the affinity of an antibody for an antigen. The present invention relates to a method for producing an antibody. The present invention relates to an antibody.

Background Art

[0002] Conventionally, a technique for controlling the affinity of an antibody for an antigen by modifying the amino acid sequence of the framework region (FR) of the antibody while maintaining the amino acid sequence of the complementarity-determining region (CDR) has been known. For example, Non-Patent Document 1 describes that amino acid residues at positions 68, 74, and 76 in the heavy-chain FR3 of a single-chain antibody (scFv) that binds to a basic epitope of troponin I were changed to aspartic acid residues, which are acidic amino acids. Non-Patent Document 1 intends to improve the affinity for troponin I by utilizing the electrostatic attraction generated by the introduction of charged amino acid residues into FR3. Patent Document 1 describes that the affinity of an antibody for an antigen can be controlled by making at least three amino acid residues in the FR3 of the light chain into charged amino acid residues. In Patent Document 1, the amino acid sequence of the FR of the antibody is modified according to the electrical properties based on the amino acid sequence of the CDR of the antibody, rather than the surface charge of the antigen.

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Non-Patent Documents

[0004]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] An object of the present invention is to provide a novel method for improving the affinity of an antibody for an antigen by modifying amino acid residues in the FR of the heavy chain without changing the amino acid sequence of the CDR, and a novel antibody having an improved affinity for an antigen.

Means for Solving the Problems

[0006] The present invention provides a method for improving the affinity of an antibody for an antigen, which comprises, in an antibody, making at least three amino acid residues of FR3 defined by the Kabat method arginine residues or lysine residues, thereby improving the affinity for the antigen as compared with the antibody before at least three amino acid residues are made arginine residues or lysine residues, and at least three amino acid residues include at least three selected from the amino acid residues at positions 68, 70, 72, 74, 75, 77, 79, 81, 82A, 82B, 83, 84, 85 and 87 of the heavy chain defined by the Kabat method, and provides a method for improving the affinity of an antibody for an antigen.

[0007] The present invention includes a step of generating an antibody in which at least three amino acid residues of FR3 defined by the Kabat method are arginine residues or lysine residues, and a step of recovering the antibody generated in that step, and the affinity of the recovered antibody for the antigen is higher than that of an antibody in which at least three amino acid residues are amino acid residues other than arginine residues and lysine residues, and at least three amino acid residues include at least three selected from the amino acid residues at positions 68, 70, 72, 74, 75, 77, 79, 81, 82A, 82B, 83, 84, 85, and 87 of the heavy chain defined by the Kabat method, and provides a method for producing an antibody.

[0008] The present invention provides an antibody in which at least three amino acid residues of FR3 defined by the Kabat method are arginine residues or lysine residues, at least three amino acid residues include at least three selected from the amino acid residues at positions 68, 70, 72, 74, 75, 77, 79, 81, 82A, 82B, 83, 84, 85, and 87 of the heavy chain defined by the Kabat method, and the affinity for the antigen is higher than that of an antibody in which at least three amino acid residues are amino acid residues other than arginine residues and lysine residues.

Advantages of the Invention

[0009] According to the present invention, an antibody with improved affinity for an antigen is provided.

Brief Description of the Drawings

[0010]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Mode for Carrying Out the Invention

[0011] In the method for improving the affinity of the antibody of the present embodiment (hereinafter also simply referred to as "method"), among the amino acid residues at positions 68, 70, 72, 74, 75, 77, 79, 81, 82A, 82B, 83, 84, 85 and 87 of the heavy chain defined by the Kabat method in the amino acid sequence of the antibody, at least three amino acid residues are arginine residues or lysine residues. Thereby, the affinity of the antibody for the antigen is improved as compared with the antibody before at least the above three amino acid residues are arginine residues or lysine residues. The amino acid residues at positions 68, 70, 72, 74, 75, 77, 79, 81, 82A, 82B, 83, 84, 85 and 87 of the heavy chain defined by the Kabat method are amino acid residues in the heavy chain FR3 of the antibody.

[0012] FR is a region other than the CDR present in the variable regions of the light and heavy chains of the antibody. FR plays the role of a scaffold connecting the three CDRs and contributes to the structural stability of the CDRs. Therefore, the amino acid sequence of FR is highly conserved among antibodies of the same species. Each of the variable regions of the light and heavy chains has three CDRs, CDR1, CDR2 and CDR3, and four FRs, FR1, FR2, FR3 and FR4. These are arranged in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 from the N-terminal side of the variable region.

[0013] In the art, a method of numbering amino acid residues of CDRs (hereinafter also referred to as "numbering method") for defining the boundaries and lengths of CDRs is known. When the amino acid residues of CDRs are numbered by the numbering method, the amino acid residues of FRs are also numbered. Examples of the numbering method include the Kabat method (Kabat EA. et al., Sequences of Proteins of Immunological Interest., NIH publication No.91-3242), the Chothia method (Chothia C. and Lesk AM., Canonical Structures for the Hypervariable Regions of Immunoglobulins., J Mol Biol., vol.196, pp.901-917, 1987), the IMGT method (Lefranc MP. et al., Developmental and Comparative Immunology, vol.29, pp.185-203, 2005), the Honergger method (Honegger A. et al., Yet Another Numbering Scheme for Immunoglobulin Variable Domains: An Automatic Modeling and Analysis Tool., J Mol Biol., vol.309, pp.657-670, 2001), the ABM method, the Contact method, and the like.

[0014] In this embodiment, the FR of the antibody may be defined by any numbering method, but is preferably defined by the Kabat method. In the definition by the Kabat method, the heavy chain FR3 of the antibody is defined as a region consisting of amino acid residues at positions 66 to 94 of the heavy chain.

[0015] In the method of this embodiment, the first antibody in which at least three amino acid residues of the heavy chain FR3 are arginine residues or lysine residues will be hereinafter also referred to as the "original antibody". As used herein, the "original antibody" means an antibody having an amino acid sequence before applying the method of this embodiment.

[0016] In the amino acid sequence of the original antibody, making at least three predetermined amino acid residues into arginine residues or lysine residues is hereinafter also referred to as "modifying" or "modification". The antibody obtained by the affinity improvement method of the present embodiment is hereinafter also referred to as "modified antibody" or "variant".

[0017] In the present embodiment, the original antibody is not particularly limited. In the method of the present embodiment, since it is not necessary to change the amino acid sequence of the CDR, the original antibody may be an antibody that recognizes any antigen. In a preferred embodiment, the original antibody is an antibody whose nucleotide sequence of the gene encoding the variable region is known or the nucleotide sequence can be confirmed. Examples of such antibodies include antibodies whose nucleotide sequences of antibody genes are disclosed in known databases, antibodies for which antibody-producing hybridomas are available, and the like. Examples of such databases include GeneBank, abYsis, IMGT, and the like.

[0018] The original antibody includes not only an antibody having a natural amino acid sequence (wild-type antibody), but also an antibody whose amino acid sequence has been artificially changed by a method other than the method for improving the affinity of the antibody of the present embodiment for the antigen. Examples of antibodies whose amino acid sequences have been artificially changed include antibodies in which the amino acid sequence of the CDR has been changed, chimeric antibodies, humanized antibodies, bispecific antibodies, single-chain antibodies (scFv) in chimeric antigen receptors, and the like.

[0019] The original antibody may be an antibody derived from any animal, and examples thereof include antibodies derived from mice, rats, hamsters, rabbits, goats, horses, chickens, humans, etc. The class of the original antibody may be any of IgG, IgA, IgM, IgD, and IgE, but is preferably IgG. The original antibody may be an antibody fragment as long as it has a variable region of the heavy chain. Examples of such antibody fragments include Fab, Fab', F(ab')2, Fd, Fd', Fv, scFv, domain antibody (dAb), reduced IgG (rIgG), diabody, triabody, etc. Among them, Fab is particularly preferred. In this specification, "antibody" is a concept that includes "antibody fragment".

[0020] As an example of the original antibody, the amino acid sequences of the light chain and heavy chain of the humanized anti-HER2 antibody (trastuzumab), and the amino acid sequence of the heavy chain of Fab are shown in Table 1. In Table 1, the underlined part indicates the variable region, and the gray-marked part indicates the CDR.

[0021]

Table 1

[0022] The amino acid sequences of each CDR and variable region of the light chain of the wild-type humanized anti-HER2 antibody are as follows. · Light chain CDR1: RASQDVNTAVA (SEQ ID NO: 4) · Light chain CDR2: SASFLYS (SEQ ID NO: 5) · Light chain CDR3: QQHYTTPPT (SEQ ID NO: 6) · Variable region: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTV (SEQ ID NO: 7)

[0023] The amino acid sequences of each CDR and variable region of the heavy chain of the wild-type humanized anti-HER2 antibody are as follows. ·Heavy chain CDR1: DTYIH (SEQ ID NO: 8) ·Heavy chain CDR2: RIYPTNGYTRYADSVKG (SEQ ID NO: 9) ·Heavy chain CDR3: WGGDGFYAMDY (SEQ ID NO: 10) ·Variable region: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (SEQ ID NO: 11)

[0024] Since the CDRs are involved in the specificity of the antibody, in the method of this embodiment, it is preferable not to change the amino acid sequences of the CDRs. That is, the amino acid sequences of the CDRs of the modified antibody are preferably the same as those of the CDRs of the original antibody.

[0025] In this embodiment, the affinity of the modified antibody for the antigen may be evaluated by kinetic parameters in the antigen-antibody reaction, or may be evaluated by immunological measurement methods such as ELISA. Examples of kinetic parameters include dissociation constant (K d ), association rate constant (k on ) and dissociation rate constant (k off ). Among them, K d is preferable. The kinetic parameters in the antigen-antibody reaction can be obtained by surface plasmon resonance (SPR) technology or the like. The value of K d in the antigen-antibody reaction of the modified antibody is, for example, about 1 / 2, about 1 / 3, about 1 / 4, about 1 / 5, about 1 / 10, about 1 / 20, about 1 / 50, about 1 / 100 or about 1 / 1000 compared to the original antibody.

[0026] In the original antibody, the amino acid residues at positions 68, 70, 72, 74, 75, 77, 79, 81, 82A, 82B, 83, 84, 85 and 87 of the heavy chain defined by the Kabat method have the following characteristics (a), (b) and (c): (a) In the amino acid sequence of the heavy chain FR3, they are amino acid residues other than the Vernier zone residues; (b) in the amino acid sequence of the heavy chain FR3, at positions where the sum of the amino acid frequencies of serine (S), threonine (T), aspartic acid (D) and glutamic acid (E) is 15% or more; and (c) in the amino acid sequence of the heavy chain FR3, an amino acid residue having a solvent-exposed surface area ratio of 10% or more.

[0027] A "vernier zone residue" is an amino acid residue among the amino acid residues contained in the FR that contributes to the structural stability of the CDR. "Position" refers to the position of an amino acid residue in a certain amino acid sequence. The position of each amino acid residue in the amino acid sequence of the FR is indicated by a number assigned by the numbering method.

[0028] "Amino acid frequency", also called amino acid occurrence frequency, refers to the ratio indicating how often a predetermined amino acid appears at each position in these amino acid sequences when multiple amino acid sequences are aligned. Amino acid frequency itself is a known index. Alignment of amino acid sequences means aligning multiple amino acid sequences so that they can be compared. Alignment of amino acid sequences can be performed by, for example, known multiple alignment programs such as ClustalW and TREBMAL. Also, the method for calculating amino acid frequency itself is known and can be calculated by the above-mentioned multiple alignment programs. Alignment of amino acid sequences and calculation of amino acid frequency can also be performed by abYsis of a public database that provides the amino acid sequence of the antibody. For example, when a certain amino acid appears in all of the multiple aligned amino acid sequences at a predetermined position, the amino acid frequency of the amino acid at that position is 100%. When a certain amino acid appears in half of the multiple aligned amino acid sequences at a predetermined position, the amino acid frequency of the amino acid at that position is 50%. When a certain amino acid does not appear at all in a predetermined position of the multiple aligned amino acid sequences, the amino acid frequency of the amino acid at that position is 0%.

[0029] When calculating the amino acid frequencies in the amino acid sequence of the heavy chain of an antibody, as a plurality of amino acid sequences, the amino acid sequences of the heavy chains of a plurality of reference antibodies are obtained. Then, the amino acid sequences of the heavy chains of the plurality of reference antibodies are aligned so that the numbers of amino acid residues in the FRs assigned by a predetermined numbering method match among the amino acid sequences of the heavy chains of the plurality of reference antibodies. The sum of the amino acid frequencies of S, T, D, and E in the amino acid sequence of heavy chain FR3 can be calculated as in the reference example described later.

[0030] The "solvent-exposed surface area" is defined in the art as the locus surface of the center of a probe sphere (1.4 Å) assuming water molecules as it rolls along the surface (van der Waals surface) of a protein molecule. The solvent-exposed surface area itself is a known index. The solvent-exposed surface area of a protein can be obtained from the three-dimensional structure data of the protein by a known program or software such as SURFace, GETAREA, or Discovery Studio. Also, the solvent-exposed surface area of each amino acid residue in a protein can be obtained. The solvent-exposed surface area of an amino acid residue in a protein varies depending on the size of the side chain of the amino acid. Therefore, the "solvent-exposed surface area ratio" is used as an index in which the solvent-exposed surface area of an amino acid residue in a protein is normalized by the size of the side chain of the amino acid. The solvent-exposed surface area ratio itself is a known index. The solvent-exposed surface area ratio in the amino acid sequence of heavy chain FR3 can be calculated as in the reference example described later.

[0031] In the present embodiment, the number of amino acid residues to be modified in heavy chain FR3 is, for example, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14.

[0032] In the modified antibody, all of the amino acid residues after modification from the amino acid residues of the original antibody may be arginine residues, or all may be lysine residues. Alternatively, some of the amino acid residues after modification from the amino acid residues of the original antibody may be arginine residues and the rest may be lysine residues.

[0033] In this embodiment, the amino acid residues at positions 68, 70, 72, 74, 75, 77, 79, 81, 82A, 82B, 83, 84, 85, and 87 of the heavy chain defined by the Kabat method in the original antibody are preferably other than arginine residues and lysine residues. The amino acid residues in the original antibody can be, for example, neutral amino acid residues, acidic amino acid residues, or histidine residues. Neutral amino acid residues are alanine residues, asparagine residues, isoleucine residues, glycine residues, glutamine residues, cysteine residues, threonine residues, serine residues, tyrosine residues, phenylalanine residues, proline residues, valine residues, methionine residues, leucine residues, and tryptophan residues. Acidic amino acid residues are aspartic acid residues and glutamic acid residues.

[0034] Means for modifying amino acid residues include substitution and insertion of amino acid residues. In the modification by substitution of amino acid residues, at least three amino acid residues selected from positions 68, 70, 72, 74, 75, 77, 79, 81, 82A, 82B, 83, 84, 85, and 87 of the heavy chain defined by the Kabat method are substituted with arginine residues or lysine residues from amino acid residues other than arginine residues and lysine residues. As a result, the above-mentioned at least three amino acid residues become arginine residues or lysine residues.

[0035] In the modification by insertion of amino acid residues, at least three arginine residues or lysine residues are inserted into the amino acid sequence of the original antibody so that they are located at at least three positions selected from positions 68, 70, 72, 74, 75, 77, 79, 81, 82A, 82B, 83, 84, 85 and 87 of the heavy chain defined by the Kabat method. For example, if you want to make positions 68, 70 and 72 of the heavy chain defined by the Kabat method into arginine residues, insert arginine residues between the amino acid residue at position 67 and the amino acid residue at position 68, between the amino acid residue at position 68 and the amino acid residue at position 69, and between the amino acid residue at position 69 and the amino acid residue at position 70 of the heavy chain of the original antibody. Thereby, each of the three inserted arginine residues is located at positions 68, 70 and 72 in the modified antibody.

[0036] In the original antibody, if any of the amino acid residues at positions 68, 70, 72, 74, 75, 77, 79, 81, 82A, 82B, 83, 84, 85 and 87 of the heavy chain defined by the Kabat method is an arginine residue or a lysine residue, the amino acid residue may be left as it is. In this case, at least three may be selected from the amino acid residues other than arginine residues and lysine residues among the amino acid residues at positions 68, 70, 72, 74, 75, 77, 79, 81, 82A, 82B, 83, 84, 85 and 87 of the heavy chain.

[0037] In one embodiment, at least three amino acid residues that are arginine residues or lysine residues in the heavy chain FR3 may include at least one selected from the amino acid residues at positions 75, 77, 79, 81, 82A, 82B, 83, 84, 85, and 87 of the heavy chain defined by the Kabat method. For example, the at least three amino acid residues may include at least two selected from the amino acid residues at positions 68, 70, 72, and 74 of the heavy chain defined by the Kabat method and at least one selected from the amino acid residues at positions 75, 77, 79, 81, 82A, 82B, 83, 84, 85, and 87 of the heavy chain defined by the Kabat method. Alternatively, the at least three amino acid residues may include at least one selected from the amino acid residues at positions 68, 70, 72, and 74 of the heavy chain defined by the Kabat method and at least two selected from the amino acid residues at positions 75, 77, 79, 81, 82A, 82B, 83, 84, 85, and 87 of the heavy chain defined by the Kabat method. Alternatively, the at least three amino acid residues may include at least three selected from the amino acid residues at positions 75, 77, 79, 81, 82A, 82B, 83, 84, 85, and 87 of the heavy chain defined by the Kabat method.

[0038] In one embodiment, at least three amino acid residues that are arginine residues or lysine residues in the heavy chain FR3 may include the amino acid residue at position 72 of the heavy chain defined by the Kabat method. For example, the at least three amino acid residues may include the amino acid residue at position 72 of the heavy chain defined by the Kabat method and at least two selected from the amino acid residues at positions 68, 70, 74, 75, 77, 79, 81, 82A, 82B, 83, 84, 85, and 87 of the heavy chain defined by the Kabat method. Alternatively, the at least three amino acid residues may include the amino acid residue at position 72 of the heavy chain defined by the Kabat method and at least two selected from the amino acid residues at positions 75, 77, 79, 81, 82A, 82B, 83, 84, 85, and 87 of the heavy chain defined by the Kabat method.

[0039] In one embodiment, at least three amino acid residues that are arginine residues or lysine residues in the heavy chain FR3 may be any of the following 1) to 6), for example. 1) The amino acid residues at positions 68, 70, and 72 of the heavy chain defined by the Kabat method; 2) The amino acid residues at positions 72, 77, and 79 of the heavy chain defined by the Kabat method; 3) The amino acid residues at positions 74, 75, and 77 of the heavy chain defined by the Kabat method; 4) The amino acid residues at positions 79, 81, and 82A of the heavy chain defined by the Kabat method; 5) The amino acid residues at positions 82B, 83, and 84 of the heavy chain defined by the Kabat method; and 6) The amino acid residues at positions 84, 85, and 87 of the heavy chain defined by the Kabat method.

[0040] In a further embodiment, in the original antibody, at least three amino acid residues of the heavy chain FR3 defined by the Kabat method may be arginine residues or lysine residues, and further, at least three amino acid residues of the light chain FR defined by the Kabat method may be arginine residues or lysine residues. Thereby, not only can the affinity for the antigen be improved compared to the original antibody, but it can also be improved compared to an antibody in which only the heavy chain FR3 is modified. Such amino acid residues of the light chain FR include at least three selected from the amino acid residues at positions 1, 3, 5, 7, 9, 10, 12, 14, 17, 18, 20, 22, 60, 63, 65, 67, 69, 70, 72, 74, 76, 77, 79, and 81 of the light chain defined by the Kabat method. The modification of the light chain FR can be carried out in the same manner as the modification of the heavy chain FR3.

[0041] In the definition by the Kabat method, FR1 of the light chain is defined as the region consisting of amino acid residues at positions 1 to 23 of the light chain, and FR3 of the light chain is defined as the region consisting of amino acid residues at positions 57 to 88 of the light chain. That is, the amino acid residues at positions 1, 3, 5, 7, 9, 10, 12, 14, 17, 18, 20, and 22 of the light chain defined by the Kabat method are in light chain FR1, and the amino acid residues at positions 60, 63, 65, 67, 69, 70, 72, 74, 76, 77, 79, and 81 of the light chain defined by the Kabat method are in light chain FR3.

[0042] In this embodiment, the number of amino acid residues modified in the light chain FR is, for example, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24. When modifying at least 3 amino acid residues in light chain FR1, the number of amino acid residues to be modified is, for example, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. When modifying at least 3 amino acid residues in light chain FR3, the number of amino acid residues to be modified is, for example, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12.

[0043] In one embodiment, in the original antibody, at least 3 amino acid residues of heavy chain FR3 defined by the Kabat method are arginine residues or lysine residues, and at least 3 amino acid residues of light chain FR3 defined by the Kabat method are arginine residues or lysine residues. In this case, at least 3 amino acid residues of light chain FR3 include at least 3 selected from the amino acid residues at positions 60, 63, 65, 67, 69, 70, 72, 74, 76, 77, 79, and 81 of the light chain defined by the Kabat method. Preferably, at least 3 amino acid residues that are arginine residues or lysine residues in light chain FR3 include the amino acid residues at positions 63, 65, and 67 of the light chain defined by the Kabat method.

[0044] In one embodiment, a method for improving the affinity of an antibody for an antigen includes, in the antibody, making at least three amino acid residues of the heavy-chain FR3 defined by the Kabat method arginine residues or lysine residues, and making at least three amino acid residues of the light-chain FR defined by the Kabat method arginine residues or lysine residues, thereby improving the affinity for the antigen as compared to the antibody before at least three amino acid residues in each of the heavy-chain FR3 and the light-chain FR are made arginine residues or lysine residues. At least three amino acid residues of the heavy-chain FR3 include at least three selected from the amino acid residues at positions 68, 70, 72, 74, 75, 77, 79, 81, 82A, 82B, 83, 84, 85, and 87 of the heavy chain defined by the Kabat method, and at least three amino acid residues of the light-chain FR include at least three selected from the amino acid residues at positions 1, 3, 5, 7, 9, 10, 12, 14, 17, 18, 20, 22, 60, 63, 65, 67, 69, 70, 72, 74, 76, 77, 79, and 81 of the light chain defined by the Kabat method.

[0045] In one embodiment, a method for improving the affinity of an antibody for an antigen includes, in the antibody, making at least three amino acid residues of the heavy-chain FR3 defined by the Kabat method arginine residues or lysine residues, and making at least three amino acid residues of the light-chain FR3 defined by the Kabat method arginine residues or lysine residues, thereby improving the affinity for the antigen as compared to the antibody before at least three amino acid residues in each of the heavy-chain FR3 and the light-chain FR3 are made arginine residues or lysine residues. At least three amino acid residues of the heavy-chain FR3 include at least three selected from the amino acid residues at positions 68, 70, 72, 74, 75, 77, 79, 81, 82A, 82B, 83, 84, 85, and 87 of the heavy chain defined by the Kabat method, and at least three amino acid residues of the light-chain FR3 include at least three selected from the amino acid residues at positions 60, 63, 65, 67, 69, 70, 72, 74, 76, 77, 79, and 81 of the light chain defined by the Kabat method.

[0046] In one embodiment, at least three amino acid residues each being an arginine residue or a lysine residue in each of the heavy chain FR3 and the light chain FR3 may be any of the following 7) to 12), for example. 7) The amino acid residues at positions 68, 70, and 72 of the heavy chain defined by the Kabat method and the amino acid residues at positions 63, 65, and 67 of the light chain defined by the Kabat method; 8) The amino acid residues at positions 72, 77, and 79 of the heavy chain defined by the Kabat method and the amino acid residues at positions 63, 65, and 67 of the light chain defined by the Kabat method; 9) The amino acid residues at positions 74, 75, and 77 of the heavy chain defined by the Kabat method and the amino acid residues at positions 63, 65, and 67 of the light chain defined by the Kabat method; 10) The amino acid residues at positions 79, 81, and 82A of the heavy chain defined by the Kabat method and the amino acid residues at positions 63, 65, and 67 of the light chain defined by the Kabat method; 11) The amino acid residues at positions 82B, 83, and 84 of the heavy chain defined by the Kabat method and the amino acid residues at positions 63, 65, and 67 of the light chain defined by the Kabat method; and 12) The amino acid residues at positions 84, 85, and 87 of the heavy chain defined by the Kabat method and the amino acid residues at positions 63, 65, and 67 of the light chain defined by the Kabat method.

[0047] In this embodiment, when the electrical property of the CDR based on the amino acid sequence of the original antibody is neutral or negatively charged, at least three amino acid residues of the heavy chain FR3, or at least three amino acid residues of each of the heavy chain FR3 and the light chain FR may be arginine residues or lysine residues. The "electrical property of the CDR" is an index uniquely defined by the present inventors and is determined based on the number of basic amino acid residues and acidic amino acid residues in the amino acid sequence of the CDR. Basic amino acid residues are lysine residues, arginine residues, and histidine residues. Specifically, the electrical property of the CDR is determined by the following formula (I).

[0048] Z = [the number of basic amino acid residues in the amino acid sequence of the CDR] - [the number of acidic amino acid residues in the amino acid sequence of the CDR] ···(I) (When Z is -1, 0, or 1, the electrical property of the CDR is neutral, when Z is 2 or more, the electrical property of the CDR is a positive charge, and when Z is -2 or less, the electrical property of the CDR is a negative charge.)

[0049] The electrical property of the CDR may be determined based on the amino acid sequence of the CDR of the light chain and / or heavy chain. When determining the electrical property of the CDR of the light chain, the amino acid sequence of the CDR in formula (I) refers to all the amino acid sequences of CDR1, CDR2, and CDR3 of the light chain. When determining the electrical property of the CDR of the heavy chain, the amino acid sequence of the CDR in formula (I) refers to all the amino acid sequences of CDR1, CDR2, and CDR3 of the heavy chain. Preferably, it is determined based on the amino acid sequences of the CDRs of both the light chain and the heavy chain. In this case, the amino acid sequence of the CDR in the above formula (I) refers to all the amino acid sequences of CDR1, CDR2, and CDR3 of the light chain and CDR1, CDR2, and CDR3 of the heavy chain. In the present embodiment, in the original antibody in which the electrical property determined based on the amino acid sequences of the CDRs of both the light chain and the heavy chain is neutral or a negative charge, the above at least 3 amino acid residues may be arginine residues or lysine residues.)

[0050] The amino acid sequence of the CDR can be obtained from a public database that discloses the sequence of the antibody gene. Alternatively, if there is a hybridoma that produces the original antibody, the amino acid sequence of the CDR can be obtained by obtaining the nucleic acids encoding the heavy chain and the light chain from the hybridoma by a known method and sequencing the nucleotide sequence of the nucleic acid.)

[0051] The electrical properties of CDRs vary depending on the antibody. For example, when defining CDRs by the Kabat method, the electrical properties of the CDRs of the wild-type anti-lysozyme antibody (HyHEL-10) used in the examples described below are negatively charged (X = -3). Also, the electrical properties of the CDRs of the wild-type humanized anti-HER2 antibody (trastuzumab) are neutral (X = 0). In the method of this embodiment, since the amino acid sequence of the CDR of the original antibody is not changed, the electrical properties of the CDR of the modified antibody are the same as those of the original antibody.

[0052] In the method of this embodiment, since at least three amino acid residues of the heavy-chain FR3 of the antibody are arginine residues or lysine residues, which are basic amino acid residues, it is considered that a positive charge is imparted to the antigen-binding site of the modified antibody. On the other hand, the mechanism by which the affinity of the modified antibody for the antigen is improved compared to the original antibody is considered to be not substantially related to the electrostatic interaction between the antigen-binding site of the antibody and the antigen. For example, lysozyme is known to be a protein with an isoelectric point (PI) of about 10. That is, lysozyme is a positively charged antigen. Then, when the method of this embodiment is applied to the wild-type anti-lysozyme antibody to modify the heavy-chain FR3, since the number of basic amino acid residues in the variable region increases, it is considered that the affinity of the modified antibody for lysozyme rather decreases. However, as shown in the examples described below, all variants of the anti-lysozyme antibody have an improved affinity for lysozyme. Thus, it is considered that the improvement of the affinity of the antibody for the antigen by the method of this embodiment does not depend on the charge of the antigen.

[0053] In this embodiment, the amino acid residues of the original antibody can be converted into arginine residues or lysine residues using known methods such as DNA recombination technology and other molecular biology techniques. Specifically, first, a polynucleotide encoding the amino acid sequence of the original antibody is obtained, and from this polynucleotide, a polynucleotide encoding the amino acid sequence of the modified antibody is prepared. Then, using the prepared polynucleotide, a modified antibody is generated by a protein expression system. The protein expression system may be an expression system using host cells or a cell-free protein synthesis system. Examples of host cells include mammalian cells, insect cells, Escherichia coli, and yeast. Examples of cell-free protein synthesis systems include a wheat germ-derived synthesis system, an Escherichia coli-derived synthesis system, and a reconstituted cell-free protein synthesis system.

[0054] For example, when there is a hybridoma that produces the original antibody, a modified antibody can be obtained as follows. First, using the RNA extracted from the hybridoma, polynucleotides encoding the heavy chain and the light chain of the original antibody are synthesized by reverse transcription reaction and RACE (Rapid Amplification of cDNA ends) method. Next, using the polynucleotide encoding the heavy chain as a template and amplifying it by PCR method using a primer for modifying at least three amino acid residues of heavy chain FR3, a polynucleotide encoding a heavy chain with modified FR3 is obtained. The obtained polynucleotide and the polynucleotide encoding the light chain of the original antibody are incorporated into a known expression vector to obtain an expression vector containing a polynucleotide encoding a modified antibody. By transforming or transfecting the obtained expression vector into an appropriate host cell, an antibody with improved affinity is generated. The generated modified antibody can be obtained by recovering it from the host cell. When modifying the light chain FR as well, a polynucleotide encoding a light chain with modified FR can be obtained by amplifying it by PCR method using a primer for modifying at least three amino acid residues of the light chain FR.

[0055] In this embodiment, the polynucleotide encoding the heavy chain and the polynucleotide encoding the light chain may be incorporated into one expression vector or separately incorporated into two expression vectors. The type of the expression vector is not particularly limited and can be determined according to the host cell. For example, expression vectors for mammalian cells, expression vectors for insect cells, expression vectors for Escherichia coli, expression vectors for yeast, etc. can be mentioned.

[0056] When obtaining a modified antibody by a cell-free protein synthesis system, a polynucleotide encoding a heavy chain with modified FR3 and a polynucleotide encoding the light chain of the original antibody are added to the cell-free protein synthesis system and incubated under appropriate conditions to obtain the modified antibody. When also modifying the light chain FR, a polynucleotide encoding a light chain with modified FR may be added instead of the polynucleotide encoding the light chain of the original antibody.

[0057] When obtaining a modified antibody that is a single-chain antibody (scFv), for example, as shown in WO2013 / 084371, using RNA extracted from a hybridoma that produces the original antibody, a polynucleotide encoding the heavy-chain variable region and a polynucleotide encoding the light-chain variable region may be synthesized by reverse transcription reaction and PCR method. These polynucleotides are ligated by methods such as overlap extension PCR to obtain a polynucleotide encoding the original antibody that is scFv. The obtained polynucleotide is amplified by PCR using a primer for modifying at least three amino acid residues of FR3 of the heavy-chain variable region to obtain a polynucleotide encoding an scFv in which FR3 of the heavy-chain variable region is modified. The obtained polynucleotide is incorporated into a known expression vector to obtain an expression vector containing a polynucleotide encoding a modified antibody that is scFv. By transforming or transfecting the obtained expression vector into an appropriate host cell, a modified antibody that is scFv can be obtained. When also modifying the light-chain FR, amplification by PCR using a primer for modifying at least three amino acid residues of the light-chain FR and a primer for modifying at least three amino acid residues of FR3 of the heavy-chain variable region may be performed to obtain a polynucleotide encoding an scFv in which FR3 of the heavy-chain variable region and FR of the light-chain variable region are modified.

[0058] In the case where there is no hybridoma producing the original antibody, antibody-producing hybridomas may be prepared by known methods such as the method described in Kohler and Milstein, Nature, vol. 256, pp. 495-497, 1975. Alternatively, RNA obtained from the spleen of an animal such as a mouse immunized with an antigen of interest may be used. When using RNA obtained from the spleen, for example, as shown in Fukunaga A and Tsumoto K, Protein Eng. Des. Sel. 2013, vol. 26, pp. 773-780, from among the polynucleotides encoding the obtained scFv, a polynucleotide encoding an scFv having a desired affinity as the original antibody may be selected by a method such as phage display method.

[0059] The method itself for recovering an antibody produced by a protein expression system is known. For example, when the antibody is produced inside a host cell, the host cell may be lysed with a solution containing an appropriate solubilizing agent to release the antibody into the solution. When the host cell secretes the produced antibody from inside the cell into the culture medium, the culture supernatant may be recovered. In a cell-free protein synthesis system, the synthesized antibody is contained in the reaction solution. The antibody released into the liquid can be recovered by a known method such as affinity chromatography. For example, when the produced antibody is IgG, it can be recovered by affinity chromatography using Protein A or G. If necessary, the recovered antibody may be purified by a known method such as gel filtration.

[0060] The modified antibody of this embodiment can be used, for example, in various tests and research, or as an active ingredient of a diagnostic agent or a therapeutic agent. The modified antibody may be used after being modified with a known substance such as a fluorescent dye, an enzyme, a radioisotope, biotin, an anticancer agent, or the like.

[0061] The antibody of this embodiment is an antibody in which at least three amino acid residues selected from the amino acid residues at positions 68, 70, 72, 74, 75, 77, 79, 81, 82A, 82B, 83, 84, 85 and 87 of the heavy chain defined by the Kabat method are arginine residues or lysine residues. The antibody of this embodiment is characterized in that the affinity for the antigen is higher than the affinity of an antibody in which at least three of the above amino acid residues are amino acid residues other than arginine residues and lysine residues. The antibody to be compared for this affinity comparison can be an antibody that recognizes the same antigen as the antibody of this embodiment.

[0062] The means for evaluating the affinity of the antibody of this embodiment for the antigen is the same as that described for the method of this embodiment above. The K value in the antigen-antibody reaction of the antibody of this embodiment d is, for example, about 1 / 2, about 1 / 3, about 1 / 4, about 1 / 5, about 1 / 10, about 1 / 20, about 1 / 50, about 1 / 100 or about 1 / 1000 as compared with an antibody in which at least three of the above amino acid residues are amino acid residues other than arginine residues and lysine residues.

[0063] The antibody of this embodiment may be an antibody that recognizes any antigen or a bispecific antibody. The antibody of this embodiment may be an antibody derived from any animal such as mouse, rat, hamster, rabbit, goat, horse, chicken, human, etc., and may be a chimeric antibody or a humanized antibody. The class of the antibody of this embodiment may be any of IgG, IgA, IgM, IgD and IgE, but IgG is preferred. The antibody of this embodiment may be an antibody fragment as long as it has a variable region of the heavy chain. Examples of such antibody fragments include Fab, Fab', F(ab')2, Fd, Fd', Fv, scFv, dAb, rIgG, diabody, triabody, etc. Among them, Fab is particularly preferred.

[0064] In the antibody of this embodiment, among the amino acid residues at positions 68, 70, 72, 74, 75, 77, 79, 81, 82A, 82B, 83, 84, 85, and 87 of the heavy chain defined by the Kabat method, the number of arginine residues or lysine residues is, for example, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14.

[0065] In the antibody of this embodiment, at least three of the above amino acid residues may be those that have become arginine residues or lysine residues from amino acid residues other than arginine residues and lysine residues by substitution or insertion of amino acid residues. In a preferred embodiment, at least three of the above amino acid residues may be those substituted from amino acid residues other than arginine residues and lysine residues to arginine residues or lysine residues.

[0066] In one embodiment, at least three amino acid residues that are arginine residues or lysine residues in the heavy chain FR3 may include at least one selected from the amino acid residues at positions 75, 77, 79, 81, 82A, 82B, 83, 84, 85, and 87 of the heavy chain defined by the Kabat method. For example, at least three amino acid residues may include at least two selected from the amino acid residues at positions 68, 70, 72, and 74 of the heavy chain defined by the Kabat method and at least one selected from the amino acid residues at positions 75, 77, 79, 81, 82A, 82B, 83, 84, 85, and 87 of the heavy chain defined by the Kabat method. Alternatively, at least three amino acid residues may include at least one selected from the amino acid residues at positions 68, 70, 72, and 74 of the heavy chain defined by the Kabat method and at least two selected from the amino acid residues at positions 75, 77, 79, 81, 82A, 82B, 83, 84, 85, and 87 of the heavy chain defined by the Kabat method. Alternatively, at least three amino acid residues may include at least three selected from the amino acid residues at positions 75, 77, 79, 81, 82A, 82B, 83, 84, 85, and 87 of the heavy chain defined by the Kabat method.

[0067] In one embodiment, at least three amino acid residues that are arginine residues or lysine residues in the heavy chain FR3 may include the amino acid residue at position 72 of the heavy chain defined by the Kabat method. For example, the at least three amino acid residues may include the amino acid residue at position 72 of the heavy chain defined by the Kabat method and at least two selected from the amino acid residues at positions 68, 70, 74, 75, 77, 79, 81, 82A, 82B, 83, 84, 85, and 87 of the heavy chain defined by the Kabat method. Alternatively, the at least three amino acid residues may include the amino acid residue at position 72 of the heavy chain defined by the Kabat method and at least two selected from the amino acid residues at positions 75, 77, 79, 81, 82A, 82B, 83, 84, 85, and 87 of the heavy chain defined by the Kabat method.

[0068] In one embodiment, the at least three amino acid residues that are arginine residues or lysine residues in the heavy chain FR3 may be, for example, any of the above 1) to 6).

[0069] In the antibody of this embodiment, at least three amino acid residues of the heavy chain FR3 defined by the Kabat method are arginine residues or lysine residues, and further, at least three amino acid residues of the light chain FR defined by the Kabat method may be arginine residues or lysine residues. Examples of such amino acid residues of the light chain FR include at least three selected from the amino acid residues at positions 1, 3, 5, 7, 9, 10, 12, 14, 17, 18, 20, 22, 60, 63, 65, 67, 69, 70, 72, 74, 76, 77, 79, and 81 of the light chain defined by the Kabat method. The antibody of this embodiment in which at least three amino acid residues of each of the heavy chain FR3 and the light chain FR are arginine residues or lysine residues has an improved affinity for the antigen as compared to the antibody of this embodiment in which at least three amino acid residues of the heavy chain FR3 are arginine residues or lysine residues.

[0070] When at least three amino acid residues of the light chain FR of the antibody of the present embodiment are arginine residues or lysine residues, among the amino acid residues at positions 1, 3, 5, 7, 9, 10, 12, 14, 17, 18, 20, 22, 60, 63, 65, 67, 69, 70, 72, 74, 76, 77, 79, and 81 of the light chain defined by the Kabat method, the number of arginine residues or lysine residues is, for example, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24. When at least three amino acid residues of the light chain FR1 are arginine residues or lysine residues, among the amino acid residues at positions 1, 3, 5, 7, 9, 10, 12, 14, 17, 18, 20, and 22 of the light chain defined by the Kabat method, the number of arginine residues or lysine residues is, for example, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. When at least three amino acid residues of the light chain FR3 are arginine residues or lysine residues, among the amino acid residues at positions 60, 63, 65, 67, 69, 70, 72, 74, 76, 77, 79, and 81 of the light chain defined by the Kabat method, the number of arginine residues or lysine residues is, for example, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12.

[0071] The antibody of the present embodiment may be an antibody in which at least three amino acid residues of the heavy chain FR3 defined by the Kabat method are arginine residues or lysine residues, and at least three amino acid residues of the light chain FR3 defined by the Kabat method are arginine residues or lysine residues. In this case, at least three amino acid residues of the light chain FR3 include at least three selected from the amino acid residues at positions 60, 63, 65, 67, 69, 70, 72, 74, 76, 77, 79, and 81 of the light chain defined by the Kabat method. Preferably, at least three amino acid residues of the light chain FR3 include the amino acid residues at positions 63, 65, and 67 of the light chain defined by the Kabat method.

[0072] The antibody of this embodiment has at least three amino acid residues of the heavy chain FR3 defined by the Kabat method being arginine residues or lysine residues, and at least three amino acid residues of the light chain FR defined by the Kabat method being arginine residues or lysine residues. At least three amino acid residues of the heavy chain FR3 include at least three selected from the amino acid residues at positions 68, 70, 72, 74, 75, 77, 79, 81, 82A, 82B, 83, 84, 85, and 87 of the heavy chain defined by the Kabat method. At least three amino acid residues of the light chain FR include at least three selected from the amino acid residues at positions 1, 3, 5, 7, 9, 10, 12, 14, 17, 18, 20, 22, 60, 63, 65, 67, 69, 70, 72, 74, 76, 77, 79, and 81 of the heavy chain defined by the Kabat method. The affinity for the antigen can be higher than that of an antibody in which at least three amino acid residues of each of the heavy chain FR3 and the light chain FR are amino acid residues other than arginine residues and lysine residues.

[0073] The antibody of this embodiment has at least three amino acid residues of the heavy chain FR3 defined by the Kabat method being arginine residues or lysine residues, and at least three amino acid residues of the light chain FR3 defined by the Kabat method being arginine residues or lysine residues. At least three amino acid residues of the heavy chain FR3 include at least three selected from the amino acid residues at positions 68, 70, 72, 74, 75, 77, 79, 81, 82A, 82B, 83, 84, 85, and 87 of the heavy chain defined by the Kabat method. At least three amino acid residues of the light chain FR include at least three selected from the amino acid residues at positions 60, 63, 65, 67, 69, 70, 72, 74, 76, 77, 79, and 81 of the heavy chain defined by the Kabat method. The affinity for the antigen can be higher than that of an antibody in which at least three amino acid residues of each of the heavy chain FR3 and the light chain FR3 are amino acid residues other than arginine residues and lysine residues.

[0074] In one embodiment, at least three amino acid residues that are arginine residues or lysine residues in each of the heavy chain FR3 and the light chain FR3 may be, for example, any of the above 7) to 12).

[0075] The antibody of this embodiment may have a neutral or negative charge in the electrical properties of the CDR based on the amino acid sequence of the CDR. The electrical properties of the CDR are determined by the above formula (I).

[0076] The antibody of this embodiment can be obtained by the method for producing the antibody of this embodiment (hereinafter also referred to as the "production method"). In the production method of this embodiment, first, an antibody is generated in which at least three amino acid residues selected from the amino acid residues at positions 68, 70, 72, 74, 75, 77, 79, 81, 82A, 82B, 83, 84, 85, and 87 of the heavy chain defined by the Kabat method are arginine residues or lysine residues. The antibody can be generated, for example, by substitution or insertion of amino acid residues in any antibody. Any antibody is preferably an antibody in which the above at least three amino acid residues are other than arginine residues and lysine residues.

[0077] In the generation of an antibody by substitution of amino acid residues, in any antibody, at least three amino acid residues selected from positions 68, 70, 72, 74, 75, 77, 79, 81, 82A, 82B, 83, 84, 85, and 87 of the heavy chain defined by the Kabat method are substituted from amino acid residues other than arginine residues and lysine residues to arginine residues or lysine residues. In the generation of an antibody by insertion of amino acid residues, at least three arginine residues or lysine residues are inserted into the amino acid sequence of any antibody so that arginine residues or lysine residues are located at at least three positions selected from positions 68, 70, 72, 74, 75, 77, 79, 81, 82A, 82B, 83, 84, 85, and 87 of the heavy chain defined by the Kabat method.

[0078] In this embodiment, for any of the above antibodies, the electrical property of the CDR based on the amino acid sequence of the CDR can be neutral or negatively charged. The electrical property of the CDR is determined by the above formula (I).

[0079] In one embodiment, at least three amino acid residues that are arginine residues or lysine residues in the heavy chain FR3 of the generated antibody may include at least one selected from the amino acid residues at positions 75, 77, 79, 81, 82A, 82B, 83, 84, 85, and 87 of the heavy chain defined by the Kabat method. Such at least three amino acid residues may include, for example, at least two selected from the amino acid residues at positions 68, 70, 72, and 74 of the heavy chain defined by the Kabat method and at least one selected from the amino acid residues at positions 75, 77, 79, 81, 82A, 82B, 83, 84, 85, and 87 of the heavy chain defined by the Kabat method. Alternatively, the at least three amino acid residues may include at least one selected from the amino acid residues at positions 68, 70, 72, and 74 of the heavy chain defined by the Kabat method and at least two selected from the amino acid residues at positions 75, 77, 79, 81, 82A, 82B, 83, 84, 85, and 87 of the heavy chain defined by the Kabat method. Alternatively, the at least three amino acid residues may include at least three selected from the amino acid residues at positions 75, 77, 79, 81, 82A, 82B, 83, 84, 85, and 87 of the heavy chain defined by the Kabat method.

[0080] In one embodiment, at least three amino acid residues that are arginine residues or lysine residues in the heavy-chain FR3 of the generated antibody may include the amino acid residue at position 72 of the heavy chain defined by the Kabat method. Such at least three amino acid residues may include, for example, the amino acid residue at position 72 of the heavy chain defined by the Kabat method and at least two selected from the amino acid residues at positions 68, 70, 74, 75, 77, 79, 81, 82A, 82B, 83, 84, 85, and 87 of the heavy chain defined by the Kabat method. Alternatively, the at least three amino acid residues may include the amino acid residue at position 72 of the heavy chain defined by the Kabat method and at least two selected from the amino acid residues at positions 75, 77, 79, 81, 82A, 82B, 83, 84, 85, and 87 of the heavy chain defined by the Kabat method.

[0081] In one embodiment, at least three amino acid residues that are arginine residues or lysine residues in the heavy-chain FR3 of the generated antibody may be, for example, any one of 1) to 6) above.

[0082] In the production method of this embodiment, in the generated antibody, at least three amino acid residues of the heavy-chain FR3 defined by the Kabat method are arginine residues or lysine residues, and further, at least three amino acid residues of the light-chain FR defined by the Kabat method may be arginine residues or lysine residues. Examples of such amino acid residues of the light-chain FR include at least three selected from the amino acid residues at positions 1, 3, 5, 7, 9, 10, 12, 14, 17, 18, 20, 22, 60, 63, 65, 67, 69, 70, 72, 74, 76, 77, 79, and 81 of the light chain defined by the Kabat method.

[0083] In the production method of the present embodiment, the antibody to be produced may be an antibody in which at least three amino acid residues of the heavy chain FR3 defined by the Kabat method are arginine residues or lysine residues, and at least three amino acid residues of the light chain FR3 defined by the Kabat method are arginine residues or lysine residues. In this case, at least three amino acid residues of the light chain FR3 include at least three selected from the amino acid residues at positions 60, 63, 65, 67, 69, 70, 72, 74, 76, 77, 79, and 81 of the light chain defined by the Kabat method. Preferably, at least three amino acid residues of the light chain FR3 include the amino acid residues at positions 63, 65, and 67 of the light chain defined by the Kabat method.

[0084] In one embodiment, the method for producing an antibody includes a step of generating an antibody in which at least three amino acid residues of the heavy chain FR3 defined by the Kabat method are arginine residues or lysine residues, and at least three amino acid residues of the light chain FR defined by the Kabat method are arginine residues or lysine residues, and a step of recovering the antibody generated in the above step. The affinity of the recovered antibody for the antigen is higher than the affinity of an antibody in which at least three amino acid residues of each of the heavy chain FR3 and the light chain FR are amino acid residues other than arginine residues and lysine residues. At least three amino acid residues of the heavy chain FR3 include at least three selected from the amino acid residues at positions 68, 70, 72, 74, 75, 77, 79, 81, 82A, 82B, 83, 84, 85, and 87 of the heavy chain defined by the Kabat method. At least three amino acid residues of the light chain FR include at least three selected from the amino acid residues at positions 1, 3, 5, 7, 9, 10, 12, 14, 17, 18, 20, 22, 60, 63, 65, 67, 69, 70, 72, 74, 76, 77, 79, and 81 of the heavy chain defined by the Kabat method.

[0085] In one embodiment, a method for producing an antibody includes a step of generating an antibody in which at least three amino acid residues of the heavy-chain FR3 defined by the Kabat method are arginine residues or lysine residues, and at least three amino acid residues of the light-chain FR3 defined by the Kabat method are arginine residues or lysine residues, and a step of recovering the antibody generated in the above step. The affinity of the recovered antibody for the antigen is higher than the affinity of an antibody in which at least three amino acid residues of each of the heavy-chain FR3 and the light-chain FR are amino acid residues other than arginine residues and lysine residues. At least three amino acid residues of the heavy-chain FR3 include at least three selected from the amino acid residues at positions 68, 70, 72, 74, 75, 77, 79, 81, 82A, 82B, 83, 84, 85, and 87 of the heavy chain defined by the Kabat method, and at least three amino acid residues of the light-chain FR include at least three selected from the amino acid residues at positions 60, 63, 65, 67, 69, 70, 72, 74, 76, 77, 79, and 81 of the heavy chain defined by the Kabat method.

[0086] In one embodiment, at least three amino acid residues that are arginine residues or lysine residues in each of the heavy-chain FR3 and the light-chain FR3 of the generated antibody may be any of 7) to 12) above, for example.

[0087] In the production method of the present embodiment, the production of the antibody can be carried out by known DNA recombination techniques and other molecular biological techniques. For example, first, a polynucleotide encoding the amino acid sequence of the antibody of the present embodiment is prepared from a polynucleotide encoding the amino acid sequence of an arbitrary antibody. Then, using the prepared polynucleotide, the antibody of the present embodiment is generated by a protein expression system. The protein expression system is the same as that described for the method of the present embodiment above.

[0088] The polynucleotide encoding the amino acid sequence of the antibody of the present embodiment can be prepared, for example, as follows, in the same manner as described in the method of the present embodiment above. Using RNA extracted from a hybridoma that produces an arbitrary antibody, a polynucleotide encoding the heavy chain of the antibody and a polynucleotide encoding the light chain are synthesized by reverse transcription reaction and RACE method. Using the polynucleotide encoding the heavy chain as a template, it is amplified by PCR using a primer for making at least three amino acid residues of heavy chain FR3 into arginine residues or lysine residues. Thereby, a polynucleotide encoding the amino acid sequence of the heavy chain of the antibody of the present embodiment can be obtained. By incorporating the obtained polynucleotide and the polynucleotide encoding the light chain of the above arbitrary antibody into an expression vector, an expression vector containing the polynucleotide encoding the antibody of the present embodiment can be obtained.

[0089] When at least three amino acid residues of each of heavy chain FR3 and light chain FR of the antibody of the present embodiment are arginine residues or lysine residues, it may be the same as the preparation of the polynucleotide encoding the heavy chain. That is, in the production method of the present embodiment, using the polynucleotide encoding the light chain of an arbitrary antibody as a template, it is amplified by PCR using a primer for making at least three amino acid residues of light chain FR into arginine residues or lysine residues, and a polynucleotide encoding the light chain of the present embodiment can be obtained.

[0090] In the production method of the present embodiment, by transforming or transfecting an appropriate host cell with an expression vector containing the polynucleotide encoding the antibody of the present embodiment, the antibody of the present embodiment can be produced in the host cell. Alternatively, the antibody of the present embodiment can be produced by adding the polynucleotide encoding the antibody of the present embodiment to a cell-free protein synthesis system and performing a synthesis reaction.

[0091] In the production method of the present embodiment, the antibody of the present embodiment can be obtained by recovering the generated antibody from the protein expression system. The method for recovering the antibody is the same as that described for the method of the present embodiment above. If necessary, the recovered antibody may be purified by a known method such as gel filtration.

[0092] Hereinafter, the present invention will be described in detail with reference to examples, but the present invention is not limited to these examples.

Examples

[0093] Example 1: Preparation of an antibody with modified amino acid residues in heavy chain FR3 Three amino acid residues of the heavy chain FR3 of the anti-lysozyme antibody were substituted with arginine residues to prepare 68, 70, 72 mutants, 79, 81, 82A mutants, and 82B, 83, 84 mutants as modified antibodies of the anti-lysozyme antibody.

[0094] (1) Acquisition of the gene of the wild-type anti-lysozyme antibody The gene synthesis of the mouse anti-lysozyme antibody (HyHEL-10) was commissioned to GenScript Japan Co., Ltd. to obtain plasmid DNA containing the gene of the wild-type anti-lysozyme antibody.

[0095] (2) Preparation of mutants of the anti-lysozyme antibody [Reagents] QIAprep Spin Miniprep Kit (QIAGEN) PrimeSTAR (registered trademark) Max DNA Polymerase (Takara Bio Inc.) Ligation high ver.2 (Toyobo Co., Ltd.) T4 Polynucleotide Kinase (Toyobo Co., Ltd.) Dpn I (Toyobo Co., Ltd.) Competent high DH5α (Toyobo Co., Ltd.)

[0096] (2.1) Primer design and PCR Based on the nucleotide sequence of the wild-type anti-lysozyme antibody gene in the plasmid DNA obtained in (1) above, a primer set for obtaining a polynucleotide encoding the light chain and a primer set for obtaining a polynucleotide encoding the heavy chain in which the following three amino acid residues in FR3 are substituted with arginine residues were designed.

[0097] - Amino acid residues at positions 68, 70, and 72 of the heavy chain defined by the Kabat method; - Amino acid residues at positions 79, 81, and 82A of the heavy chain defined by the Kabat method; and - Amino acid residues at positions 82B, 83, and 84 of the heavy chain defined by the Kabat method.

[0098] Using the plasmid DNA obtained in (1) above as a template, a PCR reaction solution with the following composition was prepared. [PCR Reaction Solution] PrimeSTAR (registered trademark) Max DNA Polymerase 12.5 μL Forward Primer (10 μM) 1 μL Reverse Primer (10 μM) 1 μL Template Plasmid (3 ng / μL) 1 μL Purified water 9.5 μL Total 25 μL

[0099] The prepared PCR reaction solution was subjected to a PCR reaction under the following reaction conditions. [Reaction Conditions] 10 seconds at 98°C, 10 seconds at 98°C, 10 seconds at 54°C, and 45 seconds at 72°C for 30 cycles, and 3 minutes at 72°C.

[0100] 1 μL of DpnI (10 U / μL) was added to the obtained PCR product (25 μL) to fragment the PCR product. Using the DpnI-treated PCR product, a ligation reaction solution with the following composition was prepared. The reaction solution was incubated at 16°C for 1 hour to perform the ligation reaction. [Ligation Reaction Solution] 2 μL of DpnI-treated PCR product 5 μL of Ligation high ver.2 1 μL of T4 polynucleotide kinase Purified water 7 μL Total: 15 μL

[0101] (2.2) Transformation, plasmid extraction and sequence confirmation The ligation reaction solution (3 μL) was added to DH5α (30 μL), and the mixture was left standing on ice for 30 minutes. Then, the mixture was heated at 42 °C for 45 seconds for heat shock. After leaving it standing on ice for 2 minutes again, the whole volume was spread on an LB plate containing ampicillin. By incubating the plate at 37 °C for 16 hours, transformants of Escherichia coli were obtained. Single colonies on the plate were picked into an LB liquid medium containing ampicillin and cultured with shaking (250 rpm) at 37 °C for 16 hours. Plasmid DNA was extracted from the obtained Escherichia coli using the QIAprep Spin Miniprep kit. The nucleotide sequences of the obtained plasmid DNAs were confirmed using pCDNA3.4 vector primers. Hereinafter, these plasmid DNAs were used as plasmid DNAs for mammalian cell expression.

[0102] (3) Expression in mammalian cells [Reagents] Expi293™ cells (Invitrogen) Expi293™ Expression Medium (Invitrogen) ExpiFectamine™ 293 Transfection Kit (Invitrogen)

[0103] (3.1) Transfection Expi293 cells were grown by shaking culture (125 rpm) at 37 °C in a 5% CO 2 atmosphere. Depending on the number of samples, 30 mL of cell culture (3.0 x 10 6Cells / mL) were prepared. Using the plasmid DNA encoding each variant and the plasmid DNA encoding the wild-type antibody, a DNA solution with the following composition was prepared and allowed to stand for 5 minutes. [DNA Solution] Amount (μL) corresponding to 15 μg of the light chain plasmid solution Amount (μL) corresponding to 15 μg of the heavy chain plasmid solution Opti-MEM (trademark) appropriate amount (mL) Total 1.5 mL

[0104] A transfection reagent with the following composition was prepared and allowed to stand for 5 minutes. ExpiFectamine reagent 80 μL Opti-MEM (trademark) 1420 μL Total 1.5 mL

[0105] The prepared DNA solution and the transfection reagent were mixed and allowed to stand for 20 minutes. The obtained mixture (3 mL) was added to the cell culture (30 mL), and then cultured with shaking at 37 °C for 20 hours (125 rpm) under a 5% CO 2 atmosphere. After 20 hours, 150 μL and 1.5 mL of ExpiFectamine™ Transfection Enhancer 1 and 2 were added to each culture, respectively, and then cultured with shaking at 37 °C for 6 days (125 rpm) under a 5% CO 2 atmosphere.

[0106] (3.2) Recovery and Purification of Antibody Each cell culture was centrifuged at 3000 rpm for 15 minutes to collect the culture supernatant. The culture supernatant contains each antibody secreted from the transfected Expi293™ cells. The obtained culture supernatant was centrifuged again at 15000×G for 10 minutes to collect the supernatant. To the obtained supernatant (30 mL), 100 μL of Ni Sepharose High Performance (GE Healthcare), a carrier for antibody purification, was added and reacted at room temperature for 2 hours. The carrier was recovered to remove the supernatant, and the carrier was washed by adding TBS (1 mL). 1000 μL of TBS containing 100 mM imidazole was added to the carrier to elute the antibody captured by the carrier. This elution operation was performed a total of 3 times to obtain an antibody solution.

[0107] Based on the nucleotide sequence of the wild-type anti-lysozyme antibody gene, the amino acid sequences of the light and heavy chains of the antibody (Fab) were determined. These amino acid sequences were as follows. Also shown below is the amino acid sequence of the heavy chain of each prepared mutant (Fab). The underlined part indicates the amino acid residue substituted from the wild-type amino acid sequence. In the amino acid sequence of the heavy chain, "CGGSHHHHHH" (SEQ ID NO: 25) at the C-terminus indicates the amino acid sequence of the His tag.

[0108] · Light chain of wild-type anti-lysozyme antibody (HyHEL-10) DIVLTQSPATLSVTPGNSVSLSCRASQSIGNNLHWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGTDFTLSINSVETEDFGMYFCQQSNSWPYTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID NO: 12)

[0109] · Heavy chain of wild-type anti-lysozyme antibody (HyHEL-10) DVQLQESGPSLVKPSQTLSLTCSVTGDSITSDYWSWIRKFPGNRLEYMGYVSYSGSTYYNPSLKSRISITRDTSKNQYYLDLNSVTTEDTATYYCANWDGDYWGQGTLVTVSAAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGSKPSICGGSHHHHHH (SEQ ID NO: 13)

[0110] · Heavy chains of 68, 70, 72 mutants of anti-lysozyme antibody DVQLQESGPSLVKPSQTLSLTCSVTGDSITSDYWSWIRKFPGNRLEYMGYVSYSGSTYYNPSLKSRI R I R R R TSKNQYYLDLNSVTTEDTATYYCANWDGDYWGQGTLVTVSAAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGSKPSICGGSHHHHHH (SEQ ID NO: 14)

[0111] · Heavy chains of 79, 81, 82A mutants of anti-lysozyme antibody DVQLQESGPSLVKPSQTLSLTCSVTGDSITSDYWSWIRKFPGNRLEYMGYVSYSGSTYYNPSLKSRISITRDTSKNQY R L R L R SVTTEDTATYYCANWDGDYWGQGTLVTVSAAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGSKPSICGGSHHHHHH (SEQ ID NO: 15)

[0112] · Heavy chains of 82B, 83, and 84 mutants of anti-lysozyme antibody DVQLQESGPSLVKPSQTLSLTCSVTGDSITSDYWSWIRKFPGNRLEYMGYVSYSGSTYYNPSLKSRISITRDTSKNQYYLDLN R V RR EDTATYYCANWDGDYWGQGTLVTVSAAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGSKPSICGGSHHHHHH (SEQ ID NO: 16)

[0113] (4) Measurement of affinity The affinity of the prepared mutants was measured using Biacore® 8K (GE Healthcare). Chicken egg white-derived lysozyme (Sigma-Aldrich) was used as the antigen for the anti-lysozyme antibody. The antigen was immobilized on a Biacore® Series S Sensor Chip CM5 (GE Healthcare) (immobilization: 50 RU). The antibody solution was diluted to prepare antibody solutions at 30 nM, 15 nM, 7.5 nM, 3.75 nM, and 1.875 nM. Each concentration of the antibody solution was fed into Biacore® 8K (GE Healthcare) (association time 120 seconds and dissociation time 1800 seconds). The measurement data were analyzed using Biacore® Evaluation software to obtain data on the affinity of each antibody.

[0114] (5) Results The K d values of each antibody are shown in Table 2. Also, the logarithm of the K d values of each antibody is shown in Figure 1. As shown in Table 2 and Figure 1, the K d values of the 68, 70, 72 mutants, 79, 81, 82A mutants, and 82B, 83, 84 mutants with modified FR3 of the heavy chain are the K dIt became lower than the value. Therefore, in these mutants, the substitution of three amino acid residues in the FR3 of the heavy chain with arginine residues improved the affinity for the antigen as compared with the wild-type antibody.

[0115]

Table 2

[0116] Reference Example: Analysis of Amino Acid Sequence of FR3 of Heavy Chain of Anti-Lysozyme Antibody In order to find the characteristics common to the mutants of Example 1 with improved affinity for the antigen, the present inventors calculated the amino acid frequencies at each position of the FR3 of the heavy chain. Further, the present inventors considered that the side chain of the amino acid residue facing the surface of the antibody molecule is involved in the improvement of the antigen affinity, and calculated the solvent-exposed surface area ratio of each amino acid residue of the FR3 of the heavy chain.

[0117] (1) Amino Acid Frequency From the abYsis of the public database, the amino acid sequences of the heavy chains of approximately 30,000 mouse antibodies were downloaded as reference antibodies. The amino acid sequences of the heavy chains of the obtained reference antibodies were aligned so that the numbers of amino acid residues in FR3 of the heavy chains assigned by the Kabat method were matched. The amino acid frequencies at each position of FR3 of the heavy chains of the obtained reference antibodies were obtained. The sequence alignment and amino acid frequencies were obtained by abYsis. At the positions corresponding to the amino acid residues modified in the mutant of Example 1, it was found that the occurrence frequencies of serine (S), threonine (T), aspartic acid (D), and glutamic acid (E) tended to be high. Therefore, the sum of the amino acid frequencies of S, T, D, and E at each position of FR3 of the heavy chain was calculated by the following formula (II). In this formula (II), the sum (X (%)) of the amino acid frequencies at a certain position in the amino acid sequences of the heavy chains of a plurality of aligned reference antibodies is calculated from the number of S, T, D, and E appearing at that position and the number of the amino acid sequences of the heavy chains of the obtained reference antibodies. Here, the numbers assigned by the Kabat method to FR3 of the heavy chains of the obtained reference antibodies were the same as those of FR3 of the heavy chain of the wild-type anti-lysozyme antibody. Therefore, the sum of the amino acid frequencies obtained from the amino acid sequences of the heavy chains of the reference antibodies was used as the value for the amino acid sequence of the heavy chain of the wild-type anti-lysozyme antibody.

[0118]

Number

[0119] (2) Solvent-exposed surface area ratio From the PDB, a public database that provides three-dimensional structural data of proteins, the amino acid sequence of the heavy chain of the wild-type anti-lysozyme antibody was searched, and the three-dimensional structural data of the heavy chain of the antibody was downloaded. Using the obtained three-dimensional structural data, the solvent-exposed surface area ratio of each amino acid residue in FR3 of the heavy chain of the wild-type anti-lysozyme antibody was obtained by Discovery Studio Client v17.2.0.16349. In Discovery Studio Client v17.2.0.16349, the solvent-exposed surface area ratio (Y(%)) was calculated by the following formula (III). In the formula, "Ala-X-Ala" is a tripeptide consisting of a sequence in which amino acid X is sandwiched between two alanines.

[0120] [Number]

[0121] (3) Results First, in Example 1, the amino acid residues to be substituted with arginine residues were selected from amino acid residues other than the residues in the Vernier zone in the heavy chain FR3 of the wild-type anti-lysozyme antibody. From (1) above, the amino acid residues substituted with arginine residues in Example 1 were amino acid residues at positions where the sum of the amino acid frequencies of S, T, D, and E was 15% or more in the amino acid sequence of the heavy chain FR3 of the wild-type anti-lysozyme antibody. Also, from (2) above, the amino acid residues substituted with arginine residues in Example 1 were amino acid residues at positions where the solvent-exposed surface area ratio was 10% or more in the amino acid sequence of the heavy chain FR3 of the wild-type anti-lysozyme antibody. Therefore, as a common feature of the mutants in Example 1, it was found that three of the amino acid residues that satisfied all of the following conditions (a), (b), and (c) were substituted with arginine residues.

[0122] (a) In the amino acid sequence of the heavy chain FR3, it is an amino acid residue other than the residues in the Vernier zone; (b) In the amino acid sequence of the heavy chain FR3, it is at a position where the sum of the amino acid frequencies of S, T, D, and E is 15% or more; and (c) It is an amino acid residue showing a solvent-exposed surface area ratio of 10% or more in the amino acid sequence of the heavy chain FR3.

[0123] In the amino acid sequence of the heavy chain FR3 of the mouse anti-lysozyme antibody, the amino acid residues having the above characteristics (a), (b), and (c) were found to be the amino acid residues at positions 68, 70, 72, 74, 75, 77, 79, 81, 82A, 82B, 83, 84, 85, and 87 of the heavy chain defined by the Kabat method.

[0124] Example 2: Preparation of an antibody with modified amino acid residues in the heavy chain FR3 (2) In antibodies other than the anti-lysozyme antibody, it was verified whether the affinity for the antigen was improved by substituting three of the amino acid residues that satisfy all of the above conditions (a), (b), and (c) with arginine residues or lysine residues. Specifically, variants of a humanized anti-HER2 antibody (trastuzumab) and a mouse anti-human insulin antibody were prepared, and the affinity of these variants for the antigen was measured.

[0125] (1) Acquisition of the gene of each antibody The gene synthesis of a humanized anti-HER2 monoclonal antibody (trastuzumab) was commissioned to GenScript Japan Co., Ltd. to obtain a plasmid DNA containing the gene of the humanized anti-HER2 antibody. A plasmid DNA containing the gene of the mouse anti-human insulin antibody was obtained in the same manner as described in the specification of US Patent Application Publication No. 2018 / 0179298.

[0126] (2) Preparation of variants (2.1) Analysis of the amino acid sequence of the heavy chain FR3 of the humanized anti-HER2 antibody From the database abYsis, the amino acid sequences of the heavy chains of approximately 30,000 human antibodies were downloaded as reference antibodies. In the same manner as in the reference example, the amino acid sequences of the heavy chains of the obtained reference antibodies were aligned, and the sum of the amino acid frequencies of S, T, D, and E at each position in the amino acid sequence of heavy chain FR3 was calculated. The solvent-exposed surface area ratio of each amino acid residue in the heavy chain FR of the humanized anti-HER2 antibody was obtained based on the three-dimensional structure data of the heavy chain of the humanized anti-HER2 antibody downloaded from the database PDB in the same manner as in the reference example. In the humanized anti-HER2 antibody as well, the amino acid residues having the above characteristics (a), (b), and (c) were the amino acid residues at positions 68, 70, 72, 74, 75, 77, 79, 81, 82A, 82B, 83, 84, 85, and 87 of the heavy chain defined by the Kabat method.

[0127] (2.2) Analysis of the Amino Acid Sequence of the Heavy Chain FR3 of Mouse Anti-Human Insulin Antibody For the sum of the amino acid frequencies of S, T, D, and E at each position in the amino acid sequence of heavy chain FR3, the data for the mouse anti-lysozyme antibody obtained in the reference example was used. The solvent-exposed surface area ratio of each amino acid residue in the heavy chain FR of the mouse anti-human insulin antibody was calculated using the modeling structure data obtained from simulations based on the amino acid sequence information of the anti-insulin antibody and the three-dimensional structure data of known mouse antibodies. The modeling was performed using Discovery Studio. In the mouse anti-insulin antibody as well, the amino acid residues having the above characteristics (a), (b), and (c) were the amino acid residues at positions 68, 70, 72, 74, 75, 77, 79, 81, 82A, 82B, 83, 84, 85, and 87 of the heavy chain defined by the Kabat method.

[0128] (2.3) Acquisition of the Gene Encoding the Heavy Chain of the Antibody Variant Based on the nucleotide sequence of the humanized anti-HER2 antibody gene in the plasmid DNA obtained in (1) above, a primer set for obtaining a polynucleotide encoding the light chain, and a primer set for obtaining a polynucleotide encoding the heavy chain in which the following three amino acid residues in FR3 are substituted with arginine residues were designed. Also, based on the nucleotide sequence of the mouse anti-insulin antibody gene in the plasmid DNA obtained in (1) above, a primer set for obtaining a polynucleotide encoding the light chain, and a primer set for obtaining a polynucleotide encoding the heavy chain in which the following three amino acid residues in FR3 are substituted with arginine residues or lysine residues were designed. Using these primer sets, PCR was carried out in the same manner as in Example 1.

[0129] - Amino acid residues at positions 68, 70, and 72 of the heavy chain defined by the Kabat method; - Amino acid residues at positions 72, 77, and 79 of the heavy chain defined by the Kabat method; - Amino acid residues at positions 74, 75, and 77 of the heavy chain defined by the Kabat method; - Amino acid residues at positions 79, 81, and 82A of the heavy chain defined by the Kabat method; - Amino acid residues at positions 82B, 83, and 84 of the heavy chain defined by the Kabat method; and - Amino acid residues at positions 84, 85, and 87 of the heavy chain defined by the Kabat method.

[0130] Using the obtained PCR products, in the same manner as in Example 1, a plasmid containing the gene encoding the mutant heavy chain and a plasmid containing the gene encoding the wild-type light chain were obtained. Using these plasmids, in the same manner as in Example 1, each antibody was expressed in Expi293 (trademark) cells. The obtained culture supernatant was purified to obtain solutions of mutants of the anti-HER2 antibody and the anti-insulin antibody.

[0131] Based on the nucleotide sequence of the wild-type humanized anti-HER2 antibody gene, the amino acid sequences of the light and heavy chains of the antibody (Fab) were determined. These amino acid sequences were as follows. Also shown below are the amino acid sequences of the heavy chains of each prepared mutant (Fab). The underlined parts indicate the amino acid residues substituted from the wild-type amino acid sequence.

[0132] · Light chain of wild-type humanized anti-HER2 antibody (trastuzumab) DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 17)

[0133] · Heavy chain of wild-type humanized anti-HER2 antibody (trastuzumab) EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPPKSCDKTGSCGGHHHHHH (SEQ ID NO: 18)

[0134] · Heavy chain of the 68, 70, 72 mutant of humanized anti-HER2 antibody EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRF R I R A RTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPPKSCDKTGSCGGHHHHHH (SEQ ID NO: 19)

[0135] · Heavy chain of 72, 77, 79 variants of humanized anti-HER2 antibody EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISA R TSKN R A R LQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPPKSCDKTGSCGGHHHHHH (SEQ ID NO: 20)

[0136] · Heavy chain of 74, 75, 77 variants of humanized anti-HER2 antibody EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADT RR N R AYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPPKSCDKTGSCGGHHHHHH (SEQ ID NO: 21)

[0137] · Heavy chain of 79, 81, 82A variants of humanized anti-HER2 antibody EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTA R L R M R SLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPPKSCDKTGSCGGHHHHHH (SEQ ID NO: 22)

[0138] · Heavy chains of 82B, 83, 84 variants of humanized anti-HER2 antibody EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMN R L RR EDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPPKSCDKTGSCGGHHHHHH (SEQ ID NO: 23)

[0139] · Heavy chains of 84, 85, 87 variants of humanized anti-HER2 antibody EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLR RR D R AVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPPKSCDKTGSCGGHHHHHH (SEQ ID NO: 24)

[0140] (3) Measurement of affinity The affinity of the prepared mutant was measured in the same manner as in Example 1 using Biacore (registered trademark) 8K (GE Healthcare). The K d values of each antibody are shown in Tables 3 and 4. Also, the logarithm of the K d values of each antibody is shown in Figures 2 and 3. In Table 4, "68, 70, 72 mutant (Lys)" and "79, 81, 82A mutant (Lys)" are mutants in which three amino acid residues in the heavy chain FR3 of the anti-insulin antibody have been substituted with lysine residues.

[0141]

Table 3

[0142]

Table 4

[0143] (4) Results As shown in Tables 3 and 4 and Figures 2 and 3, the Kd values of all mutants were lower than those of the wild-type antibody. Therefore, it was suggested that for antibodies other than the anti-lysozyme antibody, by substituting three of the amino acid residues that satisfy all of the above conditions (a), (b), and (c) with arginine residues or lysine residues, the affinity for the antigen can be improved.

[0144] Example 3: Preparation of an antibody in which both the light chain FR3 and the heavy chain FR3 are modified In the mutant in which the heavy chain FR3 was modified, the light chain FR3 was further modified to prepare a mutant in which both the light chain FR3 and the heavy chain FR3 were modified. It was verified whether the affinity of this mutant for the antigen was further improved compared to the mutant in which only the heavy chain FR3 was modified.

[0145] (1) Preparation of mutants Based on the nucleotide sequence of the gene of the humanized anti-HER2 antibody obtained in Example 2, a primer set was designed to obtain a polynucleotide encoding a light chain in which the amino acid residues at positions 63, 65, and 67 of the light chain FR3 defined by the Kabat method were substituted with arginine residues. Using this primer set, PCR was performed in the same manner as in Example 1. Using the obtained PCR product, a plasmid containing a gene encoding a light chain in which the amino acid residues at positions 63, 65, and 67 of the light chain FR3 were substituted with arginine residues was obtained in the same manner as in Example 1. As plasmids containing genes encoding the heavy chains of the humanized anti-HER2 antibody, plasmids containing genes encoding the heavy chains of the 72, 77, 79 mutants and the 82B, 83, 84 mutants obtained in Example 2 were used. Using these plasmids, each antibody was expressed in Expi293™ cells in the same manner as in Example 1. The obtained culture supernatant was purified to obtain solutions of "light chain 63, 65, 67 / heavy chain 72, 77, 79 mutant" and "light chain 63, 65, 67 / heavy chain 82B, 83, 84 mutant" as mutants of the anti-HER2 antibody in which mutations were introduced into both the light chain and the heavy chain.

[0146] (2) Measurement of Affinity The affinity of the prepared mutants was measured in the same manner as in Example 1 using Biacore® 8K (GE Healthcare). For comparison, the affinities of the wild-type humanized anti-HER2 antibody and the 72, 77, 79 mutants and 82B, 83, 84 mutants of Example 2 were also measured. The K d values of each antibody are shown in Table 5. Also, the logarithm of the K d values of each antibody is shown in Figure 4.

[0147]

Table 5

[0148] (4) Results As shown in Table 5 and Figure 4, the Kd values of all mutants were lower than those of the wild-type antibody. The Kd of the light chain 63, 65, 67 / heavy chain 72, 77, 79 mutant was lower than that of the heavy chain 72, 77, 79 mutant in which only the heavy chain FR3 was modified. Similarly, the Kd of the light chain 63, 65, 67 / heavy chain 82B, 83, 84 mutant was lower than that of the heavy chain 82B, 83, 84 mutant in which only the heavy chain FR3 was modified. Therefore, it was suggested that by modifying both the light chain FR3 and heavy chain FR3 of the antibody, an antibody with improved affinity for the antigen could be obtained compared to an antibody in which only the heavy chain FR3 was modified.

[0149] Example 4: Thermal Stability of Modified Antibodies It was examined how the thermal stability of the mutants of the anti-HER2 antibody prepared in Example 2 changed compared to the wild type.

[0150] (1) Purification of Antibodies by Size Exclusion Chromatography (SEC) Solutions containing the wild-type humanized anti-HER2 antibody and the 82B, 83, 84 mutants of the anti-HER2 antibody obtained in Example 2 were purified by SEC using AKTA (GE Healthcare). The conditions for SEC were as follows.

[0151] [SEC Conditions] Column: Superdex 200 increase 10 / 300 GL (GE Healthcare) Mobile Phase: Phosphate Buffered Saline (PBS) Flow Rate: 0.75 mL / min Elution Volume: 1 CV Column Washing: 1 CV

[0152] (2) Measurement of the Denaturation Temperature (Tm) by Differential Scanning Calorimetry (DSC) Fractions containing each antibody were diluted with PBS to prepare antibody-containing samples (final concentration 5 μM). The Tm of each antibody was measured using a MicroCal VP-Capillary DSC (Malvern Instruments Ltd). The measurement conditions were as follows.

[0153] [DSC measurement conditions] Sample volume: 400 μL Measurement range: 30 °C to 90 °C Heating rate: 60 °C / hour

[0154] (3) Results The Tm values and analysis peaks obtained by DSC measurement are shown in Table 6 and Figure 5, respectively.

Table 6

[0155] The 82B, 83, and 84 variants of the anti-HER2 antibody had a 0.46 °C decrease in thermal stability compared to the wild-type antibody, but the decrease rate was less than 1%. It was found that the thermal stability of the variants of the humanized anti-HER2 antibody hardly changed compared to the wild-type antibody.

Claims

1. A method for improving the affinity of an antibody for an antigen, comprising: the antibody is a humanized anti-HER2 antibody comprising a light chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 4, a light chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 5, and a light chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 6, and a heavy chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 8, a heavy chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 9, and a heavy chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 10; in the antibody, by making at least three amino acid residues of the framework region 3 (FR3) defined by the Kabat method arginine residues or lysine residues, improving the affinity for the antigen as compared to the antibody before the at least three amino acid residues are made arginine residues or lysine residues; A method for improving the affinity of an antibody for an antigen, wherein the at least three amino acid residues include at least three selected from the amino acid residues at positions 68, 70, 72, 77, 79, 84, 85 and 87 of the heavy chain defined by the Kabat method.

2. The method according to claim 1, wherein in the antibody, the at least three amino acid residues are made arginine residues or lysine residues by substituting the at least three amino acid residues from amino acid residues other than arginine residues and lysine residues with arginine residues or lysine residues.

3. The method according to claim 1 or 2, wherein in the antibody, the at least three amino acid residues are made arginine residues by substituting the at least three amino acid residues from amino acid residues other than arginine residues and lysine residues with arginine residues.

4. A step of generating an antibody in which at least three amino acid residues of the framework region 3 (FR3) defined by the Kabat method are arginine residues or lysine residues; A step of recovering the antibody generated in the above step; and the antibody is a humanized anti-HER2 antibody comprising a light chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 4, a light chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 5, and a light chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 6, and a heavy chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 8, a heavy chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 9, and a heavy chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 10; the affinity of the recovered antibody for the antigen is higher than the affinity of the antibody in which the at least three amino acid residues are amino acid residues other than arginine residues and lysine residues. A method for producing an antibody, wherein at least three of the at least three amino acid residues are selected from the amino acid residues at positions 68, 70, 72, 77, 79, 84, 85 and 87 of the heavy chain defined by the Kabat method. **Claim 5** The method according to claim 4, wherein the generating step comprises substituting the at least three amino acid residues from amino acid residues other than arginine residues and lysine residues with arginine residues or lysine residues. **Claim 6** The method according to claim 4 or 5, wherein the generating step comprises substituting the at least three amino acid residues from amino acid residues other than arginine residues and lysine residues with arginine residues. **Claim 7** The method according to any one of claims 4 to 6, wherein in the generating step, the antibody is generated by a protein expression system using a polynucleotide encoding the amino acid sequence of the antibody. **Claim 8** The method according to claim 7, wherein in the generating step, the antibody is generated by a host cell into which an expression vector containing the polynucleotide has been introduced. **Claim 9** The at least three amino acid residues are - amino acid residues at positions 68, 70 and 72 of the heavy chain; - amino acid residues at positions 72, 77 and 79 of the heavy chain; and - amino acid residues at positions 84, 85 and 87 of the heavy chain The method according to any one of claims 1 to 8, which is any one of the above. **Claim 10** The method according to any one of claims 1 to 9, wherein the antibody is an antibody fragment. **Claim 11** The method according to claim 10, wherein the antibody fragment is Fab. **Claim 12** A humanized anti-HER2 antibody comprising a light chain CDR1, a light chain CDR2 and a light chain CDR3 consisting of the amino acid sequences represented by SEQ ID NOs: 4, 5 and 6, respectively, and a heavy chain CDR1, a heavy chain CDR2 and a heavy chain CDR3 consisting of the amino acid sequences represented by SEQ ID NOs: 8, 9 and 10, respectively, wherein at least three amino acid residues of the framework region 3 (FR3) defined by the Kabat method are arginine residues or lysine residues, the at least three amino acid residues include at least three selected from the amino acid residues at positions 68, 70, 72, 77, 79, 84, 85 and 87 of the heavy chain defined by the Kabat method, and the affinity for the antigen is higher than the affinity of an antibody in which the at least three amino acid residues are amino acid residues other than arginine residues and lysine residues. **Claim 13** The at least three amino acid residues are - the amino acid residues at positions 68, 70 and 72 of the heavy chain; - the amino acid residues at positions 72, 77 and 79 of the heavy chain; and - the amino acid residues at positions 84, 85 and 87 of the heavy chain The antibody according to claim 12, which is any one of the above. **Claim 14** The antibody according to claim 12 or 13, wherein the at least three amino acid residues are substituted with an arginine residue or a lysine residue from an amino acid residue other than an arginine residue and a lysine residue. **Claim 15** The antibody according to any one of claims 12 to 14, wherein the at least three amino acid residues are substituted with an arginine residue from an amino acid residue other than an arginine residue and a lysine residue. **Claim 16** The antibody according to any one of claims 12 to 15, which is an antibody fragment. **Claim 17** The antibody according to claim 16, wherein the antibody fragment is Fab.

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